Mononuclear non-heme iron enzymes are involved in a wide range of reactions with oxygen including dioxygenation, hydroxylation, desaturation, 4-electron oxidation and H-atom abstraction. These enzymes are involved in the biosynthesis of antibiotics (penicillins, cephalosporins, vancomycin) and lactamase inhibitors (clavulanic acid), associated with genetic diseases (phenylketonuria, alkaptonuria, tyrosinemia, Refsum's disease), involved in the biosynthesis of lekotrienes and lipoxins (lipoxygenase (LO)), important in the bioremediation of pollutants including PCBs, and in the treatment of certain cancers (Bleomycin). These enzymes have been much more difficult to study than heme systems as they do not have the dominant spectroscopic features of the porphyrin and mostly activate dioxygen from a high-spin ferrous site which is a non-Kramers ion and generally not EPR active. New methods have been developed for the study of this wide class of enzymes including variable-temperature variable-field magnetic circular dichroism (VTVH MCD) of non-Kramers ions, VTVH MCD/zero field splitting/density functional theory of Kramers ions to define electronic structure of oxygen intermediates, and L-edge X-ray absorption spectroscopy (XAS) to define the covalencies of the metal orbitals. Application of these methods has thus far determined a general mechanistic strategy utilized by many members of this class of enzymes, defined the nature of the highly covalent Tyr-Fe III bond in the intradiol dioxygenases, and steps in the mechanism of H-atom abstraction by LO, determined the electronic structure of activated bleomycin (involved in H-atom abstraction from DNA) and shown its reactivity to be fundamentally different from that of heme systems, and developed insight into its reaction coordinate. These studies are now focused on: 1) Extending the VTVH MCD studies to define structure/function correlations for a wide range of members of this class of enzymes and their mutants related to genetic diseases; 2) understanding O2 reaction coordinates of the ferrous sites with different co-factors to generate reactive oxygen intermediates; 3) trapping and determining the geometric and electronic structures of these oxygen intermediates and their differences in reactivity (hydroxylation, desaturation, etc.); and 4) utilizing the L-edge XAS method to define electronic structure differences between heme and non-heme iron sites which correlate with differences in O2 activation.